Interleukin-7 (IL-7) is an essential pleiotrophic cytokine regulating numerous immunological events. Normal IL-7 signaling responses trigger the development, proliferation, and homeostasis of T and B cells as well as remodeling of the extracellular matrix. IL-7 activates its signaling cascade by interacting with its own cell surface receptor, IL-7R alpha, and the common gamma chain receptor through a cytokine-induced receptor heterodimerization mechanism. Under- and over-stimulation of the IL-7 pathway has been implicated in the pathogenesis of a form of severe combined immunodeficiency, autoimmune conditions, coronary artery disease, and several cancers. Structural, energetic, and cell biological studies of IL-7-induced receptor heterodimerization require a careful analysis of two distinct events: the association of IL-7 to IL-7R alpha and of IL-7:IL-7R alpha complex to the common gamma chain receptor. This research proposal will focus on the initiation step, the binding interaction of IL-7 with its alpha receptor. Results show that glycosylation of the IL- 7R alpha confers optimal binding affinity to IL-7. The importance of glycosylation of the IL-7R alpha appears to be a novel binding recognition mechanism for the common gamma chain receptor family. The thermodynamic and kinetic properties of the wild-type IL-7:IL-7R alpha interaction will be ascertained using a variety of biophysical methods including isothermal titration calorimetry and surface plasmon resonance. The functional epitopes on both IL-7 and IL-7R alpha will be determined using site-directed scanning mutagenesis, biophysical methods, and cell proliferation assays. Three-dimensional structures of free IL-7, free unglycosylated and glycosylated IL-7R alpha, and complexes of IL-7 bound to unglycosylated and glycosylated IL-7R alpha will be determined using x-ray crystallography or NMR spectroscopy. Our understanding of the binding determinants for the IL-7-receptor interaction will be used to engineer novel peptide and protein antagonists using rational protein design and phage display techniques. The long-term goal of this research involves development of a structural energetic model to explain the normal and aberrant IL-7 signaling mechanisms and the generation of new therapeutic peptides/proteins to treat the many deficiencies and diseases associated with impaired IL-7 signaling. Public Health Relevance Statement: This research proposal will focus on the structural, energetic, and cell biological studies of the interaction between human interleukin-7 and its receptor, interleukin-7 receptor alpha. Our understanding of the binding determinants for the IL-7-receptor interaction will be used to engineer novel peptide and protein antagonists using rational protein design and phage display techniques. The long-term goal of this research involves development of a structural energetic model to explain the normal and aberrant IL-7 signaling mechanisms and the generation of new therapeutic peptides/proteins to treat the many deficiencies and diseases associated with impaired human IL-7 signaling.